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Abstract

Thyroid cancer is the most common type of malignant endocrine tumor diagnosed. Previous studies have indicated that gene therapy is the most promising and effective therapeutic method for thyroid cancer. Therefore, in the present study, Na131I/5‑fluorocytosine (5‑FC) treatment was combined with cytosine deaminase (CD, encoded by the CDA gene) and sodium iodide symporter (NIS, encoded by the SLC5A5 gene) to act together as a therapeutic tool for thyroid cancer. The present study explored the combined cytotoxic effects of adenovirus‑mediated CD and NIS under the control of the progression elevated gene‑3 (PEG‑3) promoter (Ad‑PEG‑3‑CD‑NIS) with Na131I/5‑FC against the human thyroid cancer TT cell line in vitro. The PEG‑3 fragment was obtained by polymerase chain reaction (PCR) using rat genomic DNA as the template, and then Ad‑PEG‑3‑CDA‑SLC5A5 was constructed using XbaI. TT cells were transfected by recombinant adenovirus. The method of reverse transcription‑quantitative PCR was performed to test the expression of CD and NIS at the level of transcription. The morphological change was assessed by fluorescence microscopy and investigated by western blot analysis. An MTT assay was used to determine the number of living cells inhibited by single or combination therapies on TT cells. The results indicated that the PEG‑3 was successfully cloned, and was also positively regulated in 293 cells. CDA and SLC5A5 genes were highly expressed in TT cells. Na131I combined with 5‑FC significantly decreased the human thyroid cancer cells. In conclusion, combination therapy of Ad‑PEG3‑CDA‑SLC5A5 and Na131I/5‑FC induces significantly more apoptotic characteristics than either single treatment with Ad‑PEG‑3‑CDA‑SLC5A5 or Na131I/5‑FC, and low doses of Ad‑PEG‑3‑CDA‑SLC5A5 enhanced the cytotoxic effects.

Introduction

Thyroid cancer was the most common malignant
endocrine tumor diagnosed in 2006 in the USA (1,2). Thyroid
cancer is also the seventh most common type of cancer in Canadians,
and there were ~5,650 cases of thyroid cancer diagnosed in 2012
(1,2).
Concurrently, equal trends in the increase in incidence rate have
been identified all over the world (3–14). The
age-standardized incidence rate of thyroid cancer has increased
from 1.1/100,000 to 6.1/100,000 for males, and from 3.3/100,000 to
22.2/100,000 for females, from 1970 to 1972 in the USA (1,15). A
previous study indicated that the thyroid cancer incidence rate in
Canada was the fastest increasing rate in the world, t trends in
the incidence rate of thyroid cancer have demonstrated a 6.8%
increase for males and 6.9% increase for females per annum between
1998 and 2007 (16–18). Most recently, the number of new cases
of thyroid cancer is estimated to be 12.9 per 100,000 men and women
annually in 2015 in the US (19,20).

At present, previous studies (17,19,21) have
suggested that gene therapy is the most promising and effective
therapeutic method for thyroid cancer. The principle of gene
therapy depends on the intracellular conversion of a relatively
non-toxic pro-drug (or drug gene) to a toxic drug (therapeutic
protein) through gene transcription and translation processes. The
gene therapy method exhibits more advantages than conventional
chemotherapy, as it limits the pro-drug-induced toxicity to the
targeted cells (17,19,21–23). The
surrounding cells and tissues are not affected by systemic
toxicity. In previous years, the cytosine deaminase (CD) and
sodium iodide symporter (NIS) genes have been employed as
therapeutic genes in certain studies. Bentires-Alj et al
(24) investigated the feasibility of
CDA suicide gene therapy in a model of peritoneal
carcinomatosis. Kogai and Brent (23)
used the NIS gene to target cancer cells as an effective
therapeutic method. Therefore, the present study used the CD
and NIS genes to treat thyroid cancer cells.

With the exception of gene therapy, 5-fluorocytosine
(5-FC) and Na131I have also been used in cancer therapy
combined with gene therapy: Kucerova et al (25) utilized CD-mesenchymal stromal
cells/5-FC as an effective gene therapeutic tool. Zimmer et
al (26) also used
Na131I to mediate radiochemical therapy. Therefore, in
the present study, 5-FC and Na131I were combined
together to act as an assistant therapy tool for thyroid
cancer.

Following the enzyme/pro-drug systems developed and
applied in clinical practice, herpes simplex virus-1 thymidine
kinase (HSV-tk) has been used in previous years. HSV-tk is an
enzyme that may convert pro-drugs to toxic products in targeted
cells (21). In the absence of the
drug, constitutive expression of the HSV-tk gene does not
exert any harmful effects on normal cell growth. A previous study
has also suggested that transgenic animals transfected with the
HSV-tk gene have not suffered toxicity effects (21). A minimal promoter region may be
located in the progression elevated gene-3 (PEG-3), which is
associated with malignant transformation and tumor progression
(26). PEG-3 may initiate the
expression of other genes in tumor cells (27,28).
Therefore, in the present study, the PEG-3 gene was used as
the promoter for CDA and SLC5A5 gene expression in
tumor cells.

Adenovirus infection

On the day prior to viral infection, TT cells
(3.6×105 cells/well) were plated in each well of 6-well
plates. When the cells reached 70–90% confluence, the culture
medium was aspirated and the cell monolayer was washed with
pre-warmed sterile PBS.

One-Step SYBR® PrimeScript™ RT-PCR kit II
was purchased from Clontech Laboratories, Inc., (Mountainview, CA,
US). Total RNA was isolated from cultured cells using an RNAiso
Plus kit (1 ml/5×106 cells; Takara Bio, Inc.). The
concentration and purity of RNA were detected by an ultraviolet
spectrometer. cDNA was generated according to the One-Step
SYBR® PrimeScript™ RT-PCR kit II protocol. CDA
fragments were amplified with forward primer,
5′-GGAAAACGGGAAAGTTGCATCA-3′ and reverse primer,
5′-GCCTTCTCCCGCTTAGAGAC-3′. Primers for the qPCR of the mouse
SLC5A5 gene were: Forward, 5′-AGCAGGCTTAGCTGTATCCC-3′ and
reverse, 5′-AGCCCCGTAGTAGAGATAGGAG-3′, to yield 235-bp products.
Primers for the reference gene, rat β-actin, were as follows:
Forward 5′-ATCTGGCACCACACCTTC-3′ and reverse
5′-AGCCAGGTCCAGACGCA-3′. DNA amplification was conducted in a
PerkinElmer thermocycler 2400 (PerkinElmer, Inc., Waltham, MA, USA)
using an initial denaturation step at 95°C for 8 min, followed by
30 cycles of amplification with denaturation at 95°C for 30 sec,
annealing at 58°C for 30 sec, and extension at 72°C for 30 sec,
ending with a final extension at 72°C for 7 min. The
2−ΔΔCq method was used to quantify the expression levels
(30).

MTT assay

MTT assay was performed to evaluate the cell
viability in culture. The cells were seeded onto a 96-well plate at
a concentration of 1.0×105 cells/ml and a volume of 90
µl/well. Different concentrations of adenovirus
(2×105-1×106 PFU/ml) were applied to culture
wells in triplicate. Dimethyl sulfoxide was used as a negative
control. Following incubation at 37°C with 5% CO2 for 48
h, a mixture of 0.1 ml phenazine methosulfate and MTT (5 mg/ml) was
added to each well with a volume of 50 µl. The plates were
additionally incubated at 37°C for 2 h to allow MTT formazan
production. The absorbance was determined with an ELISA reader
(Thermo Fisher Scientific, Inc.) at a test wavelength of 450 nm and
a reference wavelength of 690 nm.

Statistical analysis

Statistical analyses were performed using SPSS
v.16.0 software (SPSS, Inc., Chicago, IL, USA). Values were
reported as the mean ± standard deviation. Kruskal-Wallis tests
followed by Mann-Whitney U tests were used to determine the
statistical significance of the data. P<0.05 was considered to
indicate a statistically significant difference.

Results

PEG-3 gene cloning and determination
of multiplicity of infection (MOI) in 293 cells

pSB539 is highly homologous to the
PEG-3 promoter (1,835 bp), which targets cancer cell lines
(26,27). To verify the cloning of the
PEG-3 gene and the transfection efficiency of
Ad-PEG-3 vector in 293 cells, the PEG-3 gene was
amplified by PCR, and the uptake of Ad-PEG-3 vector was
detected by fluorescence microscopy following transfection. The PCR
results indicated that PEG-3 mRNA was successfully cloned
into the Ad-vector, which was also transfected into the 293 cells
(Fig. 2A). The results of microscopy
observation demonstrated highly efficient transfection when the
virus was diluted to a MOI of 105 (~1×106
cells/ml with virus at a MOI of 5; Fig.
2B).

CD and NIS proteins express highly in
TT cells

From the results of Fig.
2, it was identified that the PEG-3 gene had been
successfully expressed in TT cells, which may trigger the positive
expression of downstream genes such as CDA and
SLC5A5. Western blot analyses were performed and the results
demonstrated that there were differences in CD and NIS protein
expression levels in TT cells when they were co-cultured with
different cell lines (PHH, Hep3B, HuH7 or CCLP1; Fig. 3).

The effect of Ad-PEG-3 vector transfection on
human thyroid living cells was determined by MTT assay. The number
of living cells was calculated as 1- the optical density reading at
600 nm. The MTT assay results indicated that either
Na131I or 5-FC could inhibit TT living cells
significantly at 24, 48, 72 or 96 h when treated with different
combinations (Table I and Fig. 4). Particularly, the Na131I
combined with 5-FC group exhibited a significantly decreased number
of living cells compared with that of the Na131I and
5-FC single treatment groups (P<0.05 and P<0.01,
respectively; Fig. 4A). Concurrently,
the living cell numbers for untransfected TT cells, used as the
control in the present study, were also significantly decreased
when treated with Na131I and 5-FC in combination
compared with that of the Na131I and 5-FC single
treatment groups (P<0.05 and P<0.01, respectively; Fig. 4B).

Table I.

Examination of the percentage of
living cells in transfected and untransfected TT cells treated with
Na131I and 5-FC.

Table I.

Examination of the percentage of
living cells in transfected and untransfected TT cells treated with
Na131I and 5-FC.

Percentage of
living cell in transfected TT cells

Percentage of
living cell in untransfected TT cells

Treatment

24 h

48 h

72 h

96 h

24 h

48 h

72 h

96 h

Na131I+5-FC

(KBq/ml +
µg/ml)

3,700+5.0

7.7±0.4

23.2±3.5

23.2±3.5

79.1±6.1

2.5±1.8

3.5±1.5

6.4±4.3

7.9±4.9

370+0.5

6.2±1.8

14.5±2.7

35.1±4.8

47.2±7.1

1.7±0.8

3.2±1.6

5.1±3.5

5.1±4.1

37+0.05

3.4±1.2

7.9±3.1

18.7±3.3

35.4±6.2

1.7±1.6

3.2±1.9

4.1±3.5

5.2±2.8

3.7+0.005

1.1±0.4

3.8±2.8

11.8±4.5

20.1±3.8

1.8±0.7

2.8±1.2

2.9±1.8

3.3±1.7

Na131I
(KBq/ml)

3,700

5.2±0.8

11.8±2.2

30.1±5.6

41.2±4.7

1.7±0.6

1.7±0.8

5.5±2.9

6.1±3.5

370

2.7±1.0

3.3±1.1

8.8±2.7

19.7±3.8

0.8±0.3

1.0±0.5

5.0±1.3

4.1±2.8

37

1.6±0.8

1.4±0.5

4.2±2.4

8.7±3.1

0.7±0.5

3.0±2.1

4.5±1.8

4.1±1.4

3.7

0.6±0.5

1.5±0.8

2.5±1.3

4.3±0.2

1.1±0.4

1.8±0.9

2.3±0.8

2.3±0.8

5-FC (µg/ml)

5.0

3.5±0.5

8.6±1.2

25.2±4.0

32.3±5.8

2.1±0.9

2.0±1.1

3.6±2.0

3.7±3.1

0.5

1.5±0.6

2.5±1.5

7.2±2.3

11.2±2.9

3.0±2.1

2.5±1.1

2.9±1.2

3.9±1.6

0.05

1.6±0.9

2.6±1.2

2.3±1.8

3.9±1.8

2.3±0.9

2.5±0.4

2.9±1.8

3.8±2.4

0.005

1.4±0.6

2.7±2.2

3.5±2.0

3.6±1.8

1.2±0.5

3.8±2.2

2.8±1.4

2.9±1.6

[i] 5-FC,
5-fluorocytosine.

Discussion

At present, the most significant problem for cancer
gene therapy is the delivery of the therapeutic gene to the
targeted tumor cells or tissues (17,21).
Indeed, almost all clinical trials currently being performed depend
on direct intra-tumor injection of the vector (27). In order to overcome this problem,
scientists have created certain vectors such as engineered
adenoviral vectors and cationic liposomes (14,15).
However, some vectors are not able to be expressed in various types
of human cancer (31). In the present
study, the pAV-murine cytomegalovirus-GFP-3FLAG vector was used to
transport the therapeutic genes. A previous study indicated that
NIS expression is primarily controlled by the thyroid-selective
transcription factors paired box gene 8 (Pax-8) and NK2 homeobox 1
(Nkx2.1) in thyroid cancer (31).
Pax-8 and Nkx2.1 target the NIS upstream enhancer through the
cardiac homeobox transcription factor Nkx2 (16,32).

The limitation of the present study was that only
one thyroid cancer cell line, the TT cell line, was employed, which
may be not sufficient to support the function of a gene as part of
a gene therapy cancer study. Therefore, in following studies, the
same in vitro experiments of the present study should be
attempted with different thyroid cancer cell lines.

To conclude, transfection with an Ad-PEG-3
plasmid into human thyroid cancer cells may inhibit tumor growth
in vitro. This may be a useful tool for gene therapy in
human thyroid cancer and other types of cancer.

Acknowledgements

The present study was supported by the National
Natural Science Foundation of China (grant no. 81072185).